Transmembrane receptors (TMRs) are proteins that span membranes and have the ability to interact with or bind a ligand, which may be a hormone, small molecule, lipid, nucleic acid, or peptide, for example. G protein-coupled receptors (GPCRs) are cell surface transmembrane proteins that translate hormone or ligand binding into intracellular signals, as do most TMRs. TMRs, or GPCRs, are found in all animals, insects, and plants. TMR, or GPCR, signaling plays a pivotal role in regulating various physiological functions including phototransduction, olfaction, neurotransmission, vascular tone, cardiac output, digestion, pain, and fluid and electrolyte balance. GPCRs, although involved in numerous physiological functions, share a number of common structural features. They contain seven membrane domains bridged by alternating intracellular and extracellular loops and an intracellular carboxyl-terminal tail of variable length.
GPCRs and other transmembrane receptors have been implicated in a number of disease states, including, but not limited to: cardiac indications such as angina pectoris, essential hypertension, myocardial infarction, supraventricular and ventricular arrhythmias, congestive heart failure, atherosclerosis, renal failure, diabetes, respiratory indications such as asthma, chronic bronchitis, bronchospasm, emphysema, airway obstruction, upper respiratory indications such as rhinitis, seasonal allergies, inflammatory disease, inflammation in response to injury, rheumatoid arthritis, chronic inflammatory bowel disease, glaucoma, hypergastrinemia, gastrointestinal indications such as acid/peptic disorder, erosive esophagitis, gastrointestinal hypersecretion, mastocytosis, gastrointestinal reflux, peptic ulcer, Zollinger-Ellison syndrome, pain, obesity, bulimia nervosa, depression, obsessive-compulsive disorder, organ malformations (for example, cardiac malformations), neurodegenerative diseases such as Parkinson's Disease and Alzheimer's Disease, multiple sclerosis, Epstein-Barr infection and cancer.
The magnitude of the physiological responses controlled by TMRs can be linked to the balance between TMR signaling and signal termination. The signaling of GPCRs and some other TMRs is controlled by a family of intracellular proteins called arresting. GPCRs are an example of transmembrane receptors which bind arrestin, activate signaling, and the like. Arrestins bind activated GPCRs, including those that have been agonist-activated and especially those that have been phosphorylated by G protein-coupled receptor kinases (GRKs).
Receptors, including GPCRs, have historically been targets for drug discovery and therapeutic agents because they bind ligands, hormones, and drugs with high specificity. Approximately fifty percent of the therapeutic drugs in use today target or interact directly with GPCRs. See e.g., Jurgen Drews, (2000) “Drug Discovery: A Historical Perspective,” Science 287:1960-1964.
TMRs, of which GPCRs are but one example, may be useful in the methods described herein.
TMRs and Signaling
TMRs, or GPCRs, can activate intracellular signaling. GPCRs recruit and regulate the activity of intracellular heterotrimeric G proteins. The activated receptor typically induces a conformational change in the associated G protein α-subunit leading to release of GDP followed by binding of GTP. Subsequently, the GTP-bound form of the α-subunit dissociates from the receptor as well as from the stable βγ-dimer. Both the GTP-bound α-subunit and the released βγ-dimer can modulate several cellular signaling pathways. These include, among others, stimulation or inhibition of adenylate cyclases and activation of phospholipases, as well as regulation of potassium and calcium channel activity. GPCRs also may not solely act via heterotrimeric G proteins.
Transmembrane Receptors (TMRs) and Internalization
Internalized TMRs, or GPCRs, are not responsive to agonists or ligands.
Subsequent to agonist or ligand exposure, initial steps of internalization produce attenuation of the signaling ability of the TMR or GPCR that may involve uncoupling of the GPCR from its cognate heterotrimeric G-protein. The cellular mechanism mediating initial steps of agonist-specific internalization is a two-step process in which agonist-occupied receptors are phosphorylated by a kinase, for example a GPCR kinase (GRK), and then bind an arrestin protein. TMRs, of which GPCRs are but one example, may bind an arrestin protein, and subsequently be internalized. The type III TGF-beta receptor is an example of a TMR, other than a GPCR, that binds arrestin, and undergoes subsequent internalization and signaling down-regulation (Chen et al., 2003, Science 301:1394-1397).
It is known, for example, that after agonists bind GPCRs, G-protein coupled receptor kinases (GRKS) phosphorylate intracellular domains of GPCRs. After phosphorylation, an arrestin protein associates with the GRK-phosphorylated receptor and uncouples the receptor from its cognate G protein. The interaction of the arrestin with the phosphorylated GPCR terminates GPCR signaling, initiates internalization, and produces a non-signaling receptor.
The arrestin bound to the GPCR targets the GPCR to claibrin-coated pits or other cellular machinery for endocytosis (i.e., internalization) by functioning as an adaptor protein, which links the GPCR to components of the endocytic machinery, such as adaptor protein-2 (AP-2) and clathrin. The internalized GPCRs are dephosphorylated and are recycled back to the cell surface, or are retained within the cell and degraded. The stability of the interaction of arrestin with the GPCR is one factor that dictates the rate of GPCR dephosphorylation, and recycling. The involvement ofGPCR phosphorylation and dephosphorylation in the internalization process has been exemplified in U.S. Ser. No. 09/933,844, filed Nov. 5, 2001, now U.S. Pat. No. 6,459,604, the disclosure of which is hereby incorporated by reterence in its entirety.
There is a need for methods to identify compounds that can activate TMR signaling, or GPCR signaling, with reduced internalization of the TMR. Such compounds have increased signaling with decreased signal termination.